Prevalence of Plasmodium spp. in Anopheles mosquitoes in Thailand: a systematic review and meta-analysis

Background The entomological inoculation rate (EIR) is one of the key indices used to evaluate malaria transmission and vector control interventions. One of the components of the EIR is the sporozoite rate in Anopheles vectors. A systematic review and meta-analysis was performed to identify the prevalence of Plasmodium spp. in field-collected Anopheles species across Thailand. Methods This systematic review was registered under the PROSPERO number CRD42021297255. Studies that focused on the identification of Plasmodium spp. in Anopheles mosquitoes were identified from the electronic databases PubMed, Web of Science, and Scopus. The quality of the identified studies was determined using the Strengthening the Reporting of Observational Studies in Epidemiology approach. The proportion of Anopheles mosquitoes collected, Anopheles vectors for Plasmodium species, and specificity of Anopheles vectors for Plasmodium species were analyzed. The pooled prevalence of Plasmodium species among the primary vectors (Anopheles dirus, Anopheles minimus, and Anopheles maculatus) was estimated using the random-effects model. Results Of the 1113 studies identified, 31 were included in the syntheses. Of the 100,910 Anopheles mosquitoes identified for species and sibling species, An. minimus (40.16%), An. maculatus (16.59%), and Anopheles epiroticus (9.18%) were the most prevalent Anopheles species. Of the 123,286 Anopheles mosquitoes identified, 566 (0.46%) were positive for Plasmodium species. The highest proportions of Plasmodium species were identified in Anopheles hodgkini (2/6, 33.3%), Anopheles nigerrimus (2/24, 8.33%), Anopheles balabacensis (4/84, 4.76%), An. dirus (114/4956, 2.3%), Anopheles annularis (16/852, 1.88%), Anopheles kochi (8/519, 1.54%), Anopheles vagus (3/215, 1.4%), and Anopheles baimaii (1/86, 1.16%). The pooled prevalence of Plasmodium species identified in the main Anopheles vectors was 0.4% of that of Plasmodium species identified in An. dirus was 2.1%, that of Plasmodium species identified in An. minimus was 0.4%, and that of Plasmodium species identified in An. maculatus was 0.4%. Conclusions We found a low prevalence of Plasmodium infection in Anopheles mosquitoes across Thailand. Therefore, the use of EIR to determine the impact of vector control intervention on malaria parasite transmission and elimination in Thailand must be undertaken with caution, as a large number of Anopheles specimens may be required. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-022-05397-2.

the Indochinese Peninsula in Southeast Asia. It shares a border with Myanmar, Laos, Cambodia, and Malaysia, leading to a constant migration of foreign workers and refugees across the borders [3]. The presence of efficient vectors and long rainy season in Thailand indicates that there are several areas that can serve as mosquito habitats and that mosquito-borne diseases present a life-threatening public health challenge from both indigenous and imported cases [4]. Despite ongoing efforts to combat malaria, approximately 4473 confirmed cases were reported in Thailand in 2020. Plasmodium vivax is the dominant cause of infection (4098 cases, 92%), and the number of cases involving Plasmodium falciparum is much lower (256 cases, 5.7%) but still significant [5]. In Thailand, malaria cases are reported to have a high prevalence in vulnerable forest and forest fringes along rural stretches of border areas [3,4]. Tak Province, which is a neighbor to Myanmar, had one of the highest incidences of malaria in Thailand in 2020 (1241 cases), followed by Yala Province in peninsular Thailand, on the border with Malaysia (1075 cases), and Kanchanaburi Province, another neighbor of Myanmar, with 539 confirmed cases [5]. Military personnel, forest workers, refugees, and local migrants are the individuals at the highest risk [3]. Although the malaria cases in Thailand in 2020 decreased by 24% compared to those in 2019 (5859 cases) [5], considerable effort is needed to achieve malaria elimination by 2024 [6]. In addition to techniques for the diagnosis of malaria and effective therapy, mosquito control is one of the most effective interventions against malaria. Distribution of insecticide-treated nets (ITNs) and indoor residual spraying (IRS) are currently used [5,7]. With the implementation of the National Malaria Elimination Strategy 2017-2026, ITNs have started being distributed at a ratio of one ITN per two people, with the aim of at least 90% coverage in each transmission focus. Retreatment of the net is performed regularly every 6-12 months. However, if the retreatment process is inaccessible, long-lasting insecticidal nets (LLINs) are distributed. IRS is implemented in cases where both ITNs and LLINs cannot be allocated [7]. In 2020, over 102,150 LLINs were distributed with an average of 75% coverage across Thailand [5]. Unfortunately, these methods tackle only indoor-and late-biting vectors, and indoor-resting Anopheles mosquitoes are not suitable for reducing outdoor transmission. Thus, additional approaches are needed to protect people from outdoor-and early-biting vectors [8].
To assess the transmission dynamics as well as the effectiveness of the vector control methods, it has been suggested that the entomological inoculation rate (EIR) should be evaluated annually [25]. The EIR measures the frequency of infectious bites by an Anopheles mosquito per person over time, combining the human biting rate and the sporozoite rate (SR) [26]. However, not all infectious bites result in blood-stage malaria in human hosts because Plasmodium parasites possibly experience significant bottlenecks during their sporogony cycle in mosquitoes [27]. The SR remains one of the important entomological indicators not only in assessing EIR but also in identifying Anopheles vectors that contribute to Plasmodium transmission in endemic settings [28]. Indicators other than EIR could also influence the transmission dynamics. As EIR can be estimated from the human biting rate, it has been demonstrated that very high vector density and high SR could sustain malaria transmission over a large part of the year [29]. In view of the evaluation of pre-oocyst formation blocking interventions, (e.g., gametocytocidal drugs), a previous study suggested that the oocyst formation rate is another highly reliable entomological indicator of mosquito infectiveness [30]. Although research into naturally infected Anopheles mosquito has been conducted for decades, there is a need for a comprehensive systematic review and meta-analysis focusing on the prevalence of malaria parasites in field-collected Anopheles species across Thailand. We herein mainly focused on the combined prevalence of both sporozoite and oocyst infection rates, hereby termed "Plasmodium infection. " The information collected and synthesized in this study improves our understanding of the local transmission dynamics of malaria vector species, particularly primary vectors, and may offer useful data for the evaluation of vector control interventions and malaria transmission.

Protocol
The systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines [31]. The systematic review was registered at PROS-PERO with the number CRD42021297255.

Literature search
Relevant studies were searched from three research databases, namely, PubMed, Web of Science, and Scopus, from inception to March 30, 2021. The reference lists of the included studies were also examined to ensure that relevant studies were not missed. We combined relevant search terms with Boolean operators, and the terms "(malaria OR Plasmodium) and (anopheles OR anopheline) and (Thailand OR Thai OR Siam)" were used to identify relevant studies in each database (Additional file 1: Table S1). Studies that focused on the identification of Plasmodium spp. in Anopheles mosquitoes were selected. The reference lists of the included studies were also screened to ensure that relevant studies were not missed. The search included only studies that were published in English between 1945 and 2021.

Eligibility criteria and study selection
Studies were selected using the PICO method. The elements of this method are as follows: P: Participants. The participants included in the study were Anopheles mosquitoes in Thailand. I: Intervention. No intervention was applied in the present study. C: Comparator. No comparator was used in the present study. O: Outcome. The outcome of interest was the presence of Plasmodium spp. in any stage that was identified in Anopheles mosquitoes. Thus, the inclusion criteria were composed of cross-sectional studies that identified Plasmodium spp. among Anopheles mosquitoes collected in Thailand. The exclusion criteria were studies with incomplete data for extraction, studies for which the full text was unavailable, reviews or systematic reviews, in vitro studies, papers describing the development of assays, and letters to the editor/comments/editorials. Two authors (CS and MK) independently screened the titles and abstracts and selected studies based on the eligibility criteria. First, the titles and abstracts generated by the electronic search were checked. Second, the full texts were examined, and studies that did not meet the eligibility criteria were excluded, with the reasons recorded. Any differences in study selection between the two authors were resolved by mutual consensus.

Data extraction and quality assessment
Pilot data extraction tables were used to collect information from each included study. The following information was collected: name of the first author, year of publication, study sites, season and time at which mosquitoes were collected, mosquito collection methods, mosquito species, number of mosquitoes, methods of Plasmodium detection, Plasmodium identified, and stage of Plasmodium spp. The quality of the studies included was determined using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement, which includes 22 parameters [32]. The quality of each study was assessed as high quality (> 75%), moderate quality (50-75%), or low quality (< 50%). High-quality and moderate-quality studies were included in the systematic review. Low-quality studies were excluded.

Data syntheses
The proportion of Anopheles mosquitoes collected in the studies, Anopheles vectors positive for Plasmodium species, and Plasmodium spp. identified in Anopheles mosquitoes are presented as frequencies and percentages. The pooled prevalence of Plasmodium species among Anopheles mosquitoes and primary vectors (An. dirus, An. minimus, and An. maculatus) was estimated using a random-effects meta-analysis using the DerSimonian and Laird method [33]. The proportions from each study were pooled using logit transformation, and back transformation to a proportion was performed using generalized linear mixed models (GLMMs). The individual study weights were not calculated by the GLMMs. The metaanalyses of proportion studies were conducted using the command "metaprop_one" in Stata version 14.0 software (StataCorp LLC, College Station, TX, USA) as described previously [34]. Forest plots were used to depict the study-specific proportions with 95% exact confidence intervals and overall pooled estimates with 95% Wald confidence intervals. The Chi-square statistic of the likelihood ratio test was used to identify the presence of significant heterogeneity when the P-value was less than 0.05. As the individual study weights were not calculated by the GLMMs, publication bias assessment was not performed in the present study. For the meta-analyses of proportion studies with low proportion outcomes, funnel plots were not used, as they are ineffective at detecting potential publication bias [35].

Search results
A total of 1113 candidate studies were identified through PubMed (379 studies), Web of Science (448 studies), and Scopus (286 studies). After 483 duplicates were removed, 630 studies were screened for titles and abstracts, and 207 studies remained for further full-text examination. The examination of the full text of the studies identified 19 studies [15-17, 19, 20, 23, 36-48] that met the eligibility criteria. One hundred and eighty-eight studies were excluded for specific reasons: 46 reviews, 31 mosquito identifications, 29 parasite studies, 28 assay developments, and 18 malaria biology in Anopheles mosquitoes; 11 for incomplete data; eight letters to the editor/comments/editorials; six genetic studies of malaria; three in vitro studies; three studies for which the full text was unavailable; two systematic reviews; one animal study; one case report; and one clinical trial. An additional 12 relevant studies [49][50][51][52][53][54][55][56][57][58][59][60] from the reference lists of the included studies and Google Scholar were examined for full texts. All of them met the eligibility criteria and were included in the systematic review. Overall, 31 studies [15-17, 19, 20, 23, 36-60] were included in the present systematic review (Fig. 1).

Anopheles mosquitoes collected in the studies
The list of the Anopheles mosquitoes collected from the studies is shown in

Proportion of Plasmodium species identified in each Anopheles vector
To identify where malaria transmission occurs, reliable estimations of the proportions of infective Anopheles mosquitoes, as reflected by the presence of sporozoites in the salivary gland, are needed [66,67]. Several techniques have been used to quantify sporozoites in mosquitoes. Dissection and microscopic examination of the salivary glands is considered the "gold standard" method, LR test: RE vs FE Model chi^2 = 20.3, p = 0.000) Rattanarithikul et al. [16] Baker et al. [49] Study Poolphol et al. [41] Somboon et al. [20] Upatham et al. [59] Rattaprasert et al. [42] Gingrich et al. [38] Green et al. [19] Rosenberg et al. [15] 0 but this approach is labor intensive and impractical in the field [68]. Enzyme-linked immunosorbent assay to detect circumsporozoite proteins (CSP-ELISA) is another widely used technique [69]. However, CSP-ELISA has been shown to give false positive results, thus overestimating the real SR [70]. Molecular-based methods have been developed to improve sensitivity and specificity [71]. In our analysis, 19 studies (61.30%) used CSP-ELISA as a means of Plasmodium detection. Six studies (19.35%) used dissection of the salivary gland and gut. It is possible that the limitations of the dissection and CSP-ELISA methods used in the studies could have affected the estimation of sporozoite infection. Six studies (19.35%) used polymerase chain reaction (PCR)-based techniques. Sumruayphol et al. [23] performed nested PCR and realtime PCR on 9260 An. epiroticus specimens and found only six mosquitoes infected with P. falciparum and three with P. vivax. In another study, Tainchum et al. [47] also used real-time PCR for Plasmodium detection in 1090 An. minimus specimens and found only one positive sample. These studies suggested that even using methods with high sensitivity, the prevalence of Plasmodium species in Anopheles mosquitoes was very low, suggesting that the prevalence of parasite vectors in Thailand was genuinely low. Other techniques such as rapid dipstick immunochromatographic assays (Vec-Test ™ Malaria) [72] and near-infrared spectroscopy [73] have also been developed for Plasmodium detection in Anopheles mosquitoes. Overall, to avoid the overestimation of SR and the EIR, it is highly recommended that all positive CSP-ELISA samples be reanalyzed or the results confirmed by performing Plasmodium-specific PCR [70].
Anopheles dirus had the highest pooled prevalence of Plasmodium species identified (2.1%) in our analysis. However, it should also be noted that the high prevalence of infection does not necessarily translate to the species being the main vector. Other factors also play a crucial role in the importance of primary vectors, for instance, the species must often be abundantly present and prefer to feed on humans [10]. Previous studies also reported LR test: RE vs FE Model chi^2 = 109.1, p = 0.000) Study Somboon et al. [20] Rattanarithikul et al. [16] Gingrich et al. [38] Harbach et al. [39] Rosenberg et al. [15] Sriwichai et al. [45] Sithiprasasna et al. [57] Edwards et al. [53] Coleman et al. [17] Ratanatham et al. [56] Eamkum et al. [52] Sattabongkot et al. [43] Zollner et al. [48] Tainchum et al. [47] Baker et al. [49] 0 relatively low numbers of collected An. dirus specimens (ranging from 10-78 mosquitoes/location) recently [41,42,[45][46][47]53]. Therefore, it is vital to assess transmission indicators (e.g., EIR) to determine the importance of each vector species. The prevalence of Plasmodium species identified in An. dirus decreased from 5% in 1987 [49] to 1% in 1990 [38]. However, the prevalence of Plasmodium species identified in An. dirus increased to 4% in 1990 [15] and decreased to 1-3% during 1991-2017 [16,19,20,41,42]. The yearly trend results were heterogeneous, and differences in study sites had to be considered. The pooled prevalence of Plasmodium species identified in An. minimus and An. maculatus was the same at 0.4%. These results suggest that the likelihood of finding an infected wild An. minimus or An. maculatus is lower than that of finding an infected An. dirus. The reasons for this observation remain to be investigated. However, several factors influencing vectorial capacity and competence have been documented, including mosquito longevity, the duration of sporogonic development, and the susceptibility or resistance of the vector to Plasmodium [74]. A previous study of three laboratory strains of An. dirus, An. minimus, and An. sawadwongporni showed similar susceptibility to P. vivax infection using an artificial feeding system [75]. These laboratory-raised mosquitoes are highly inbred and may be genetically dissimilar to the originally sampled population [76]. In our analysis, natural Plasmodium infection in wild Anopheles mosquitoes was also considerably different among populations and species. In another study, large differences in P. falciparum infection were observed in a wild population of Anopheles gambiae Giles in West Africa [77]. Therefore, it is not surprising to find differences in Plasmodium prevalence in the diverse field populations in Thailand. The present study had some limitations. First, the studies that were included for systematic review were not performed in all areas in which malaria cases have been reported. There are missing mosquito data from the Thailand-Malaysia border, where high levels of malaria have been reported. Hence, the systematic review did not represent the overall prevalence in Thailand. Second, the majority of the studies (14 studies, 45.16%) used in our Zollner et al. [48] Rattanarithikul et al. [16] Sriwichai et al. [44] Coleman et al. [51] Coleman et al. [17] Study Baker et al. [49] Sattabongkot et al. [43] Somboon et al. [20] Upatham et al. [59] Rosenberg et al. [15] Sumruayphol et al. [58] 0  Fig. 7 The pooled prevalence of Plasmodium spp. in An. maculatus. ES, prevalence estimate; 95% CI: confidence interval analysis used only morphological keys for species identification, and an additional 12 studies (38.71%) did not indicate which identification method was used. Therefore, some of the Anopheles specimens in these studies were only classified into complexes or groups. We therefore simply reported and analyzed the species data using the information presented in the studies. Only five studies (16.13%) used molecular techniques to confirm the species of the Anopheles specimens after the initial morphological identification into complexes or groups.
To reflect the true prevalence of Plasmodium in each Anopheles species, particularly the primary and secondary malaria vectors, species confirmation using molecular techniques should also be performed. Third, it has been demonstrated that a positive SR may coincide with peak mosquito populations [16,20,48]. Thus, the prevalence of Plasmodium infection in each mosquito species could also be varied depending on season and mosquito abundance. In our analysis, only cross-sectional studies were included and we did not attempt to factor seasonal variation, as some included studies did not report seasonal information of positive Plasmodium infection specimens. However, is it possible that in any of the included studies, data for one species might have been collected during high transmission, whereas data for other species might have been collected during low transmission seasons, which might be a source of overestimation and underestimation of the importance of different vectors. Fourth, we included studies that identified Anophelesharbored sporozoites and also oocysts of Plasmodium spp. As these stages are different indicators and may have different interpretations, we used the oocyst formation rate to study infection in the vector, that is, to show the susceptibility of the vector to infection; however, it does not indicate the importance of the vector in transmission. Therefore, the prevalence of Plasmodium spp. in Anopheles mosquitoes indicates the infection rates rather than the transmission capability. Finally, there are